用哪个版本的基因组和注释文件好？| 亲测

What Ensembl genome version should I use for alignments? (e.g. toplevel.fa vs. primary_assembly.fa)

这是一个很细节也很实际的问题，到底用哪个版本？

参考：

What Ensembl genome version should I use for alignments? (e.g. toplevel.fa vs. primary_assembly.fa)

Results differ when using different ensembl versions

 

First part options:

• dna_sm - Repeats soft-masked (converts repeat nucleotides to lowercase)
• dna_rm - Repeats masked (converts repeats to to N's)
• dna - No masking

Second part options:

• .toplevel - Includes haplotype information (not sure how aligners deal with this)

• .primary_assembly - Single reference base per position

 

大部分都推荐使用soft-mask版本的，也就是没有把repeat替换为N。

 

下载hg19基因组：http://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/

参考：基因组各种版本对应关系

从genecode下载hg19注释文件：ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_27/

UCSC也可以下载，不过只能从网页导出。http://genome.ucsc.edu/cgi-bin/hgTables

注：genecode貌似出了问题，https://www.gencodegenes.org/releases/26lift37.html，里面ebi的链接无法下载了。

参考：http://www.biotrainee.com/thread-2035-1-1.html

 

基因组不是越新越好的，看看最新的CNS，里面很少有用最新版本的基因组，为什么？因为注释没跟上，你做出来的东西可能和别人对不上。

 

亲测

用不同版本的基因组效果会怎么样？

我做了转录组的测试，用的hg19和GRCh38

结论如下：

1. reads比对到基因组上的情况大致相同，基本没有差别；

2. 用不同的注释文件，基因表达的结果差距非常大。同样都是用featureCounts

GRCh38的结果：

Assigned        306852
Unassigned_Unmapped     0
Unassigned_MappingQuality       0
Unassigned_Chimera      0
Unassigned_FragmentLength       0
Unassigned_Duplicate    0
Unassigned_MultiMapping 36280
Unassigned_Secondary    0
Unassigned_Nonjunction  0
Unassigned_NoFeatures   56950
Unassigned_Overlapping_Length   0
Unassigned_Ambiguity    19771

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file /home/lizhixin/databases/ensembl/release91/Homo_s ... ||
||    Features : 1199851                                                      ||
||    Meta-features : 58302                                                   ||
||    Chromosomes/contigs : 47                                                ||
||                                                                            ||
|| Process BAM file /home/lizhixin/project/scRNA-seq/reanalyze/first_five ... ||
||    Paired-end reads are included.                                          ||
||    Assign fragments (read pairs) to features...                            ||
||                                                                            ||
||    WARNING: reads from the same pair were found not adjacent to each       ||
||             other in the input (due to read sorting by location or         ||
||             reporting of multi-mapping read pairs).                        ||
||                                                                            ||
||    Read re-ordering is performed.                                          ||
||                                                                            ||
||    Total fragments : 419853                                                ||
||    Successfully assigned fragments : 306852 (73.1%)                        ||
||    Running time : 0.05 minutes                                             ||

　　

hg19的结果：

Assigned        586467
Unassigned_Unmapped     0
Unassigned_MappingQuality       0
Unassigned_Chimera      0
Unassigned_FragmentLength       0
Unassigned_Duplicate    0
Unassigned_MultiMapping 66997
Unassigned_Secondary    0
Unassigned_Nonjunction  0
Unassigned_NoFeatures   133437
Unassigned_Overlapping_Length   0
Unassigned_Ambiguity    47278

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file /home/lizhixin/databases/cellranger_ref/refdata-c ... ||
||    Features : 1130716                                                      ||
||    Meta-features : 32738                                                   ||
||    Chromosomes/contigs : 45                                                ||
||                                                                            ||
|| Process BAM file /home/lizhixin/project/scRNA-seq/reanalyze/first_five ... ||
||    Paired-end reads are included.                                          ||
||    Assign fragments (read pairs) to features...                            ||
||    Total fragments : 834179                                                ||
||    Successfully assigned fragments : 586467 (70.3%)                        ||
||    Running time : 0.05 minutes                                             ||

 

不同的注释文件千万不要乱用！！！